We examined the influence of insulin on S6 kinase activity in fibroblasts from skin biopsies of 5 insulin-sensitive and 5 insulin-resistant Pima Indians. Activities of S6 kinase and S6 peptide (RRLSSLRA) kinase were assayed in extracts of cells obtained at 0, 15, 30, 45 and 60 minutes of exposure to 100nM insulin or to carrier. Exposure of fibroblasts, obtained from insulin sensitive subjects, to insulin resulted in a 2.2-fold activation of S6 kinase activity measured in soluble fractions of the cells. Activity was maximally stimulated by 15 min of exposure to insulin and remained elevated through 30 min before declining. In contrast, S6 kinase was activated by only 50% in fibroblasts obtained from insulin-resistant subjects. The timecourse of activation was similar to that seen in cells from sensitive subjects. S6 peptide kinase activity was stimulated 35% over basal activity in response to insulin in cells obtained from both sensitive and resistant subjects. The activation was maximal by 15 min and declined to basal levels thereafter. These results support a genetic basis of abnormal insulin signal transduction in Pima Indians and suggest that the gene or genes responsible for insulin resistance encode a protein(s) that contributes to the mechanism of insulin action intracellularly. Because activation of S6 peptide kinase was normal in cells from resistant subjects, it is likely that the genetic lesion causing impaired insulin signal transduction may affect only some aspects of insulin action.